The intracellular parasite Toxoplasma gondii converts from fast-dividing tachyzoite stages to slowly divid- ing tissue-encysted bradyzoite stages that characterize the chronic phase of the infection. Toxoplasma encephalitis (TE) from reactivation of these semi-dormant cysts remains an important cause of morbidity and mortality in AIDS patients. Current drugs are poorly tolerated, and they do not kill the bradyzoite stages. Thus, there is a critical need to identify Toxoplasma proteins that are important for bradyzoite survival inside cysts, such that novel therapeutics can be designed to target these proteins. Toxoplasma replicates within a parasitophorous vacuole (PV) surrounded by the PV membrane (PVM). To satisfy its auxotrophic requirements, host nutrients need to cross the PVM barrier, yet Toxo- plasma proteins in the PVM involved in nutrient acquisition are largely unknown. We discovered that two Toxoplasma proteins, GRA17 and GRA23, which are secreted from its dense granule secretory orga- nelles into the PV lumen, make pores in the PVM that mediate the flux of small molecules, such as nutri- ents, across the PVM. ?gra17 parasites are slow growing and avirulent in mice, consistent with certain nutrients becoming limiting to ?gra17 parasite growth. The rationale for this project is that we have re- cently shown that GRA17 is also important for survival of bradyzoites inside cysts and therefore Toxo- plasma proteins that mediate: (i) nutrient acquisition at the PVM; or (ii) trafficking of proteins involved in nutrient acquisition from the PV lumen to the PVM, likely determine survival of bradyzoites inside cysts. Recently, we performed a CRISPR/Cas9-mediated genome-wide loss-of-function screen and identified Toxoplasma genes that have a strong fitness defect in the ?nutrient sensitized? ?gra17 but not in wild-type parasites. Because ?gra17?gra23 parasites are not viable, our working hypothesis is that Toxoplasma genes involved in trafficking of GRA23 to the PVM, after its secretion into the PV lumen, are likely among the hits of our screen as are other genes mediating nutrient uptake at the PVM. What re- mains lacking, however, is confirming most hits from this screen. Therefore, in our first aim we will identi- fy hits from the CRISPR/Cas9 loss-of-function screens that are synthetically lethal/sick in ?gra17 para- sites. In our second aim we will identify Toxoplasma genes that determine survival of bradyzoites inside cysts by: (i) mediating nutrient acquisition at the PVM; or (ii) affecting correct trafficking of proteins in- volved in nutrient acquisition to the PVM. At the completion of this R21, our expected outcomes are to have identified novel Toxoplasma proteins that are important for viability of bradyzoites inside cysts. These results are expected to have an important positive impact because they will provide new targets for the rational development of therapeutics against Toxoplasma bradyzoites which currently do not exist.